PROJECT SUMMARY Though great advances have been made in uncovering molecular pathways that maintain pluripotency and regulate differentiation in vertebrate cells in culture, the control of pluripotent stem cells and their progeny in the context of whole animals is poorly understood. The long-term goal of our work is to obtain a mechanistic understanding of how pluripotent stem cells are controlled in vivo such that they can regenerate any missing cell type in an adult animal. The overall objective in this application is to identify the cellular and molecular mechanisms that control the specification, maintenance, and response to regeneration of pluripotent adult stem cells called ?neoblasts? in the acoel Hofstenia miamia. Hofstenia can regenerate any missing cell type and is amenable to high-throughput functional studies of regeneration. The rationale for choosing a new model system over planarians, the more established system, is that Hofstenia produces manipulable embryos in large numbers, allowing the use of methods currently unavailable in planarians to answer outstanding questions about neoblast biology. The experiments proposed here will combine lineage-tracing methods with functional genomic approaches to discover and characterize critical regulators of pluripotent cells. This project will ask three major questions about neoblasts: 1) What are the developmental origins of Hofstenia neoblasts and which molecular pathways are required for the formation of these pluripotent cells, 2) Which gene regulatory networks mediate the maintenance of Hofstenia neoblasts, and 3) How do individual neoblasts respond to amputation, i.e., proliferate, migrate, and/or differentiate during regeneration. Lineage tracing and in situ hybridization in Hofstenia embryos, techniques that have been established as feasible in the applicants' hands, will be utilized to identify the developmental origins of pluripotent cells. Transcriptome profiling will then be used to identify candidate genes that control the specification of neoblasts during development; the functions of these candidates will be studied by RNAi and CRISPR/Cas9-mediated genome editing. Genome-wide assays for assessing chromatin state will be used to identify regulatory DNA for known neoblast genes and the upstream transcription factors that putatively regulate the genes. The functions of the DNA and the associated transcription factors in maintenance of pluripotent stem cells will be assayed via CRISPR-Cas9 genome editing and RNAi respectively. Individual neoblasts will be labeled by photoconverting fluorescent proteins that label neoblasts and their proliferation, migration, and differentiation will be monitored via time lapse confocal microscopy. This proposal is innovative in its use of a new model system that enables the study of long-standing questions about stem cell biology by using approaches that cannot be achieved by studying established model systems. This project will reveal basic cellular and molecular principles for in vivo control of pluripotency that have the potential to inform the development of new applications in human regenerative medicine.